A composite coating for anti-Cronobacter pylori biofilm, its preparation method and application

Abstract

This invention belongs to the field of material surface modification and antibacterial technology, and relates to a composite coating for inhibiting Cronobacter pylori biofilm formation, its preparation method, and its application. This composite coating combines the self-polymerization-adhesion properties of dopamine with the biofilm-inhibiting activity of chitosan oligosaccharides to form a stable and durable functional coating on the surface of a substrate material. Its preparation includes: preparing a dopamine-chitosan oligosaccharide composite coating solution; immersing a clean substrate material in the solution; and reacting under weakly alkaline conditions to allow polydopamine and chitosan oligosaccharides to co-deposit on the substrate surface, ultimately forming the composite coating. This invention features a simple process, mild conditions, and good biocompatibility of the raw materials. The resulting coating effectively inhibits the initial adhesion of Cronobacter pylori on surfaces such as glass, stainless steel, and silicone, and significantly weakens or even prevents the formation of its biofilm. It has broad application prospects in food processing equipment, medical devices, packaging materials, underwater artifact preservation, and underwater equipment.

Description

Technical Field

[0001] This invention belongs to the field of material surface modification and microbial control technology, specifically relating to a composite coating for anti-Cronobacter biofilm, its preparation method and application. Background Technology

[0002] Kronobacter ( Cronobacter *Cronobacter spp.* is a foodborne pathogen that poses a serious threat to public health. Infants, the elderly, and those with weakened immune systems are particularly susceptible, and infection can lead to severe meningitis, sepsis, and necrotizing enterocolitis, with a high mortality rate. *Cronobacter spp.* is widely distributed in the natural environment and food processing environments and has been proven to be one of the main sources of contamination in infant formula. A key pathogenic and survival characteristic of this bacterium is its strong adhesion ability, enabling it to firmly attach to various materials such as stainless steel, glass, and silicone, and further develop into a biofilm. A biofilm is a structured community formed by bacteria to adapt to adverse environments. The bacterial cells within the biofilm are encapsulated by extracellular polymers secreted by the bacteria themselves. This physical barrier significantly increases the bacteria's resistance to conventional disinfectants and antibiotics, making it a persistent source of contamination that is difficult to completely remove from food processing equipment and medical environments, and potentially causing cross-contamination and increasing the risk of infection.

[0003] To address the challenges posed by bacterial adhesion and biofilms, current strategies primarily include the application of antimicrobial agents, physical cleaning processes, and surface modification of materials. However, these traditional methods all have limitations. For example, long-term use of chemical antimicrobial agents may lead to bacterial resistance, and their residues may pose toxicological risks and environmental pollution problems. Physical cleaning methods often fail to completely remove established biofilms, resulting in limited effectiveness. Therefore, developing a safe, efficient, and durable anti-adhesion surface coating technology is of great significance.

[0004] In recent years, inspired by the adhesive proteins of marine mussels, dopamine and its self-polymerized polydopamine coatings have attracted widespread attention. Polydopamine can form strongly adhesive nanofilms on the surfaces of almost all types of organic or inorganic materials through self-polymerization in mild, weakly alkaline aqueous solutions. This coating not only imparts a certain degree of antifouling ability to the substrate, but its abundant active groups can also serve as a platform for secondary functionalization, allowing for the grafting of other functional molecules. Chitosan oligosaccharides, as products of chitin deacetylation degradation, have better water solubility and stronger permeability compared to high molecular weight chitosan, while also exhibiting broad-spectrum antibacterial activity and good biocompatibility, showing promising applications in the field of antibacterial materials. However, how to stably and firmly fix chitosan oligosaccharides onto material surfaces remains a key technical challenge restricting its application.

[0005] Patent CN116971180A discloses a polylactic acid / dopamine / chitosan oligosaccharide hydrophilic antibacterial nonwoven fabric and its preparation method. This method constructs a polydopamine-modified layer on the surface of a polylactic acid substrate through dopamine oxidative self-polymerization, improving the substrate's hydrophilicity. Furthermore, it enhances the adhesion of chitosan oligosaccharides to the substrate surface through a cross-linking reaction between the amino groups of chitosan oligosaccharides and the quinone groups of polydopamine. However, this technology has significant limitations. First, it is only suitable for a single polylactic acid nonwoven fabric substrate, and cannot be adapted to materials such as glass commonly used in food processing equipment and medical devices, resulting in a narrow application range. Second, the step-by-step modification preparation process is cumbersome and difficult to control during production. Third, the evaluation of antibacterial performance is relatively limited, only examining the antibacterial effects against Escherichia coli and Staphylococcus aureus, without conducting research on the antibacterial effects against Cronobacter, nor quantitatively analyzing the material's surface antibacterial adhesion and biofilm inhibition effects. Biofilms are a key factor leading to persistent contamination and cross-contamination on food processing surfaces.

[0006] In summary, existing antibacterial technologies suffer from risks of chemical antibacterial agent resistance and residue, difficulty in removing biofilms through physical cleaning, challenges in immobilizing functional molecules on material surfaces, and a lack of effective strategies for preventing Cronobacter from adhering to and biofilming on commonly used materials in food processing and medical devices. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the existing technology by providing a composite coating for anti-Cronobacter biofilm, its preparation method, and its application. This anti-Cronobacter biofilm composite coating can firmly adhere to the surface of various materials, exhibits synergistic effects through dopamine and chitosan oligosaccharides, and possesses both rapid response and long-lasting inhibitory properties.

[0008] The objective of this invention can be achieved through the following technical solutions: A composite coating for anti-Cronobacter biofilms is formed by the co-deposition reaction of polydopamine (PDA) and chitosan oligosaccharide (COS) and is firmly attached to the surface of a substrate material.

[0009] Furthermore, the substrate material is selected from at least one of glass, stainless steel, and silicone, which are widely used in the food industry (such as baby bottles, pipes, and storage tanks) and medical devices.

[0010] The present invention also provides a method for preparing the composite coating of the anti-Cronobacter biofilm as described above, comprising the following steps: a. Preparation of coating solution: Dopamine hydrochloride and chitosan oligosaccharide are dissolved in a buffer solution and mixed evenly to obtain a dopamine-chitosan oligosaccharide composite coating solution; the dopamine hydrochloride is a stable salt-forming compound formed by dopamine and hydrochloric acid, and is a commercially available stable form of dopamine; b. Substrate pretreatment: The substrate material is sequentially immersed in acetone, ethanol and deionized water for ultrasonic cleaning to obtain a clean surface; c. Co-deposition reaction: Immerse the pretreated substrate material in the coating solution prepared in step a, and react for 2-24 hours under the conditions of pH 7.5-8.5 and temperature 20-40℃. d. Post-treatment: After the reaction is completed, the substrate material is removed and the surface is rinsed three times with deionized water. Then, the substrate material is placed in a vacuum drying oven and dried at 50°C for 4-5 hours to obtain the composite coating of the anti-Cronobacter biofilm on the surface of the substrate material. In this step, the cleaning is to gently rinse the surface with deionized water to remove physically adsorbed unreacted substances.

[0011] Further, in step a, the buffer solution is a Tris-HCl buffer solution with a concentration of 10 mM and a pH of 8.5.

[0012] Furthermore, the concentration of dopamine was 0.5-2.5 mg / mL; the concentration of chitosan oligosaccharide was 2.5-5.0 mg / mL.

[0013] Furthermore, in step a, the mass ratio of dopamine to chitosan oligosaccharide is (1-2):(2-5).

[0014] Furthermore, in step c, the reaction is carried out under oscillating conditions to make the deposition more uniform; preferably, the reaction system is placed on a shaker and oscillated at a speed of 100~300 rpm.

[0015] The present invention also provides an application of the composite coating for resisting Cronobacter biofilm as described in any of the preceding claims, the composite coating being used to prepare articles having anti-Cronobacter adhesion and / or anti-biofilm functions.

[0016] Furthermore, the articles include food processing equipment, food contact utensils (such as the inner walls of baby bottles and milk storage tanks), medical equipment, packaging materials, underwater cultural relic protection components, and underwater equipment.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) Rapid, durable, and efficient synergistic anti-adhesion and anti-biofilm properties: This invention combines the self-polymerization-adhesion properties of dopamine with the natural antibacterial activity of chitosan oligosaccharides to form a stable and durable antibacterial functional layer on the surface of the substrate material. The polydopamine-chitosan oligosaccharide (PDA-COS) composite coating provided by this invention has been demonstrated through systematic quantitative tests at three time points: 6 hours, 12 hours, and 24 hours. It can rapidly (6 hours) significantly reduce the initial adhesion of Cronobacterium tumefaciens on the surfaces of three typical materials: glass, silicone, and stainless steel, and continuously and effectively inhibit the accumulation of biofilm biomass over time. At all test time points, the anti-adhesion and anti-biofilm effects of this composite coating are significantly better than those of a single polydopamine coating, confirming that the introduction of chitosan oligosaccharides endows the coating with excellent synergistic antibacterial activity.

[0018] (2) Broad-spectrum substrate applicability: This invention makes full use of the universal adhesion properties of polydopamine. Through a one-step co-deposition method, a composite coating can be stably constructed on the surfaces of various materials with different properties (such as hydrophilic glass, hydrophobic silicone, and stainless steel covered with metal oxides). It has good versatility and is suitable for the modification of food contact surfaces or medical device surfaces of different materials.

[0019] (3) Safety and environmental protection: The dopamine and chitosan oligosaccharide used in this invention are both natural sources with good biocompatibility. The constructed composite coating avoids the problems of drug resistance, cytotoxicity and environmental residue that may be caused by traditional chemical antibacterial agents. It is particularly suitable for the food industry and medical field where safety requirements are high.

[0020] (4) Simple preparation process: The composite coating of the present invention is prepared by one-step co-deposition method, which is simple to operate, mild reaction conditions, and does not require complex equipment or harsh processes. It is easy to achieve large-scale production and promotion and application, and has broad application prospects in food processing equipment, medical devices, packaging materials and even underwater cultural relics protection and underwater equipment that require resistance to biofouling. Attached Figure Description

[0021] Figure 1 Comparison of scanning electron microscopy results of glass substrates with different coatings, where a is the blank glass of Comparative Example 2, b is the PDA coating of Comparative Example 1, and c is the PDA-COS composite coating of Example 1. Figure 2 Comparison of atomic force microscopy results of glass substrates with different coatings, where a is the blank glass of Comparative Example 2; b is the PDA coating of Comparative Example 1; and c is the PDA-COS composite coating of Example 1. Figure 3XPS spectra of glass substrates with different coatings, including blank glass (Comparative Example 2), PDA coating (Comparative Example 1), and PDA-COS composite coating (Example 1). Figure 4 These are O 1s energy spectrum images of glass substrates with different coatings, including blank glass (Comparative Example 2), PDA coating (Comparative Example 1), and PDA-COS composite coating (Example 1). Figure 5 Figure 1 shows the test results of antibacterial adhesion properties of different substrates modified with PDA coating and PDA-COS composite coating, as well as blank substrates. In the figure, a is glass substrate; b is 304 stainless steel sheet substrate; c is silicone sheet substrate. Figure 6 The images show the surface anti-biofilm test results of different substrates modified with PDA coating and PDA-COS composite coating, as well as a blank substrate. In the images, a is a glass substrate; b is a 304 stainless steel sheet substrate; and c is a silicone sheet substrate. Detailed Implementation

[0022] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0023] Unless otherwise specified, all raw materials used in this invention are commercially available products, such as those described in the following examples and comparative examples: Chitosan oligosaccharide and dopamine hydrochloride were purchased from Shanghai E. En Chemical Technology Co., Ltd. Crystal violet was purchased from Aladdin Biochemical Technology Co., Ltd.

[0024] This invention employs a one-step co-deposition process, which effectively mitigates the interference of chitosan oligosaccharides on the dopamine polymerization process by precisely controlling the pH, feed ratio, and reaction time of the reaction system. This allows polydopamine generation and chitosan oligosaccharide cross-linking to occur simultaneously, forming a molecular-level interpenetrating network structure. The chitosan oligosaccharides are covalently fixed in the coating network, constructing a non-leaching contact sterilization mechanism. This coating can be uniformly formed on the surface of different substrates such as glass, stainless steel, and silicone, achieving dynamic inhibition of the Cronobacter biofilm formation process at multiple time points.

[0025] Example 1 Preparation of a polydopamine-chitosan oligosaccharide (PDA-COS) composite coating on glass surfaces to create anti-Cronobacter biofilms: Chitosan oligosaccharide (COS, molecular weight ≤3000 Da) was added to 50 mL of 10 mM Tris-HCl buffer at pH 8.5 and stirred to dissolve, resulting in a solution with a chitosan oligosaccharide concentration of 5 mg / mL. Dopamine hydrochloride was then added to the solution and stirred to dissolve, bringing the concentration of dopamine hydrochloride to 2 mg / mL. The mixture was then thoroughly mixed to obtain the composite coating solution.

[0026] A glass slide (1 cm × 1 cm) that had been ultrasonically cleaned and dried in sequence with acetone, ethanol and deionized water was immersed in the above composite coating solution and placed on a shaker (speed: 200 rpm) and reacted at 25°C for 12 hours.

[0027] After the reaction was completed, the glass slide was removed and the surface was rinsed three times with deionized water. Then the glass slide was placed in a vacuum drying oven and dried at 50°C for 5 hours, thus obtaining the composite coating of the anti-Cronobacter biofilm on the surface of the glass slide.

[0028] Comparative Example 1 The same glass substrate and cleaning method as in Example 1 were used. A Tris-HCl buffer solution (10 mM, pH=8.5) containing only dopamine hydrochloride (concentration 2.0 mg / mL) was prepared as the coating solution. The cleaned glass slide was immersed in the solution and reacted at 25°C and 200 rpm for 12 hours. The subsequent cleaning and drying steps were exactly the same as in Example 1, and a single polydopamine coating sample was obtained.

[0029] Comparative Example 2 Using the same glass substrate as in Example 1, and after the same cleaning process, without any coating treatment, it served as a blank control group.

[0030] The PDA-COS composite coating obtained in Example 1, the single PDA coating obtained in Comparative Example 1, and the blank glass substrate in Comparative Example 2 were characterized by scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy to complete the surface morphology and chemical structure analysis of the samples.

[0031] Scanning electron microscopy (SEM) observation: The microstructure of the three groups of samples was observed using field emission scanning electron microscopy, and the results are shown in the figure. Figure 1 .in, Figure 1 (a) The surface of the blank glass substrate is flat and smooth, with no obvious material adhering to it; Figure 1 (b) A single PDA coating forms a regular nanoparticle morphology on its surface, with uniform particle distribution; compared to a single PDA coating, Figure 1(c) The surface structure of the PDA-COS composite coating is more compact and smooth, and the particle interface tends to be blurred, indicating that the introduction of chitosan oligosaccharide participated in the assembly process of polydopamine and formed a more uniform composite network structure.

[0032] Atomic force microscopy (AFM) analysis: The surface 3D morphology of three samples was observed using atomic force microscopy in tapping mode, and the root mean square roughness (Rq) was calculated. The results are as follows: Figure 2 As shown. Figure 2 (a) The surface height of the blank glass substrate varies very little (approximately -9.0 nm to 8.0 nm), exhibiting a flat morphology; Figure 2 (b) The surface height difference of a single PDA coating increases (from approximately -20.0 nm to 18.0 nm), resulting in a significant increase in roughness; Figure 2 (c) The surface height difference of the PDA-COS composite coating further increases (from approximately -32.0 nm to 30.0 nm), indicating that the formation of the composite coating further increases the effective specific surface area, which is beneficial to enhancing the contact efficiency between the coating and bacteria, thereby improving the antibacterial performance.

[0033] X-ray photoelectron spectroscopy (XPS) analysis: The surface elemental composition and chemical state of three samples were analyzed using X-ray photoelectron spectroscopy. The full-spectrum scan results are shown below. Figure 3 As shown, the blank glass substrate mainly exhibits characteristic peaks of elements such as Si and O; the single PDA coating shows obvious C, N, and O peaks, with the N peak originating from the amino / imine groups of polydopamine; the N peak intensity of the PDA-COS composite coating is significantly higher than that of the single PDA coating, and the peak position is slightly shifted. This is because a large number of amino groups in chitosan oligosaccharides react with the quinone groups of polydopamine to form more abundant nitrogen-containing functional groups, confirming the successful co-deposition of chitosan oligosaccharides.

[0034] Peak fitting was performed on the O 1s high-resolution energy spectrum, and the results are as follows: Figure 4 As shown, the O 1s peak of the blank glass substrate is located at approximately 532.5 eV, corresponding to the Si-O-Si structure; the O 1s peak of the single PDA coating can be fitted as two components: C=O (approximately 531.5 eV) and CO (approximately 533.0 eV); the O 1s peak of the PDA-COS composite coating shifts towards lower binding energies, and the proportion of C=O component decreases significantly while the proportion of CO component increases. This indicates that the amino groups of chitosan oligosaccharide and the quinone groups of polydopamine underwent a cross-linking reaction, consuming some of the quinone groups (C=O) and generating more hydroxyl or ether bonds (CO). This confirms at the chemical bond level that a stable covalent cross-linking network was formed between the two.

[0035] The characterization results consistently indicate that a PDA-COS composite coating was successfully prepared on the glass surface via a one-step co-deposition method. Chemical cross-linking occurred between chitosan oligosaccharide and polydopamine, forming a composite structure that is denser, more uniform, and has a higher surface roughness than a single polydopamine coating. This provides a favorable physicochemical basis for subsequent anti-Cronobacter adhesion and anti-biofilm properties.

[0036] Example 2 The preparation method of the PDA-COS composite coating for anti-Cronobacter biofilm on stainless steel surface is basically the same as that in Example 1, except that the substrate in this example is a 304 stainless steel sheet.

[0037] Comparative Example 3 The same 304 stainless steel substrate and cleaning method as in Example 2 were used. A Tris-HCl buffer solution (10 mM, pH=8.5) containing only dopamine hydrochloride (concentration 2.0 mg / mL) was prepared as the coating solution. The cleaned 304 stainless steel sheet was immersed in the solution and reacted at 25°C and 200 rpm for 12 hours. The subsequent cleaning and drying steps were exactly the same as in Example 1 to obtain a single polydopamine coating sample.

[0038] Comparative Example 4 Using the same 304 stainless steel sheet substrate as in Example 2, and after the same cleaning process, without any coating treatment, it served as a blank control group.

[0039] Example 3 A PDA-COS composite coating for preparing an anti-Cronobacter biofilm on a silicone surface was prepared using a method that was basically the same as in Example 1, except that the substrate in this example was a silicone sheet.

[0040] Comparative Example 5 The same silicone substrate and cleaning method as in Example 3 were used. A Tris-HCl buffer solution (10 mM, pH=8.5) containing only dopamine hydrochloride (concentration 2.0 mg / mL) was prepared as the coating solution. The cleaned 304 stainless steel sheet was immersed in the solution and reacted at 25°C and 200 rpm for 12 hours. The subsequent cleaning and drying steps were exactly the same as in Example 1, and a single polydopamine coating sample was obtained.

[0041] Comparative Example 6 Using the same silicone sheet substrate as in Example 3, and after the same cleaning process, without any coating treatment, it served as a blank control group.

[0042] The following tests were performed on the PDA-COS composite coatings prepared in Examples 1-3, the single polydopamine coatings prepared in Comparative Examples 1, 3, and 5, and the blank substrates in Comparative Examples 2, 4, and 6: Anti-adhesion test: Cronobacter tumefaciens frozen in preservation tubes was inoculated into fresh LB liquid medium and cultured overnight at 37°C and 200 rpm. The culture was zeroed with fresh LB liquid medium, and the OD600 of the overnight culture was adjusted to approximately 0.5 using fresh LB liquid medium. An appropriate amount of the culture was centrifuged at 4°C and 8000 rpm for 10 min. After labeling, the supernatant was discarded, and an equal volume of fresh LB liquid medium was added. The mixture was shaken to obtain a standard bacterial suspension. Samples prepared in each example and comparative example were sterilized and then immersed in 10 mL of PBS buffer for 2 h to reach equilibration. The samples were transferred to 12-well plates, and 1 mL of the standard bacterial suspension was added to each well. The plates were incubated at 37°C for 6 h, 12 h, and 24 h, respectively. After incubation, the samples were removed and gently rinsed three times with 10 mL of PBS buffer to remove any unadhered bacteria. The sample was placed in a 50 mL sterile centrifuge tube containing 5 mL of PBS buffer and sonicated at low power (40%) for 5 min to detach the adhering bacteria. The sonicated solution was appropriately diluted and spread onto LB agar plates, incubated overnight at 37°C, and colony-forming units (CFU) were counted to evaluate the antibacterial adhesion properties of the coating. All procedures were performed under aseptic conditions.

[0043] Anti-biofilm test: After sterilization, the samples prepared in each example and comparative example were immersed in 10 mL PBS buffer for 2 h to reach equilibrium. The samples were placed in 12-well plates, and 400 μL of standard bacterial suspension and 1600 μL of fresh TSB medium were added. The plates were incubated at 37℃ for 6 h, 12 h, and 24 h, respectively. Biofilm formation was determined using crystal violet staining: The samples were removed, and surface airborne bacteria were gently washed away with PBS buffer and dried at room temperature for 20 min. The samples were transferred to new 12-well plates, and 2000 μL of methanol was added to each well for fixation for 15 min. The methanol was removed, and 0.1% crystal violet solution was added for staining for 10 min. Excess staining solution was removed, and each well was repeatedly rinsed with sterile water until the eluent was colorless. 2000 μL of 95% ethanol was added to each well, and the crystal violet was dissolved by standing at room temperature for 30 min. The ethanol solution was transferred to new well plates, and the OD600 value was measured using a microplate reader to characterize the amount of biofilm formation.

[0044] Test results: 1. Glass substrate Using the blank glass of Comparative Example 2 as a control, the anti-adhesion and anti-biofilm test results of the PDA-COS composite coating of Example 1 and the PDA coating of Comparative Example 1 are as follows: Figure 5 a and Figure 6As shown in Figure a, at the three time points of 6 h, 12 h, and 24 h, the amount of adhering bacteria and biofilm in the PDA-COS coated group were significantly lower than those in the blank control group and the single PDA coated group, indicating that the PDA-COS coating has rapid and long-lasting anti-adhesion and anti-biofilm properties.

[0045] 2. Stainless steel base Using the blank stainless steel sheet of Comparative Example 4 as a control, the anti-adhesion and anti-biofilm test results of the PDA-COS composite coating of Example 2 and the PDA coating of Comparative Example 3 are as follows: Figure 5 b and Figure 6 As shown in b, the PDA-COS coating also exhibits rapid and durable anti-adhesion and anti-biofilm properties on stainless steel surfaces. At 24 h, its anti-adhesion efficiency and anti-biofilm efficiency both exceed 30%, significantly higher than that of a single PDA coating.

[0046] 3. Silicone substrate Using the blank silicone sheet of Comparative Example 6 as a control, the anti-adhesion and anti-biofilm test results of the PDA-COS composite coating of Example 3 and the PDA coating of Comparative Example 5 are as follows: Figure 5 c and Figure 6 As shown in c, the PDA-COS coating maintained good performance on the hydrophobic silica surface. At 6 h, the PDA-COS coating reduced bacterial adhesion by approximately 60% and biofilm biomass by approximately 50%; while the single PDA coating only reduced these by approximately 48% and 30%, respectively. At 12 h and 24 h, the inhibitory effect of the PDA-COS coating was still significantly better than that of the single PDA coating, indicating that the coating can effectively inhibit early bacterial adhesion and prevent long-term biofilm development.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composite coating for resisting Cronobacter cloacae biofilms, characterized in that, The composite coating is formed by the co-deposition reaction of polydopamine and chitosan oligosaccharide and adheres to the surface of the substrate material.

2. The composite coating for an anti-Cronobacter biofilm according to claim 1, characterized in that, The substrate material is selected from at least one of glass, stainless steel, and silicone.

3. A method for preparing a composite coating for an anti-Cronobacter biofilm as described in claim 1 or 2, characterized in that, Includes the following steps: a. Preparation of coating solution: Dissolve dopamine hydrochloride and chitosan oligosaccharide in buffer solution and mix evenly to obtain dopamine hydrochloride-chitosan oligosaccharide composite coating solution; b. Substrate pretreatment: Cleaning the substrate material; c. Co-deposition reaction: Immerse the pretreated substrate material in the coating solution prepared in step a, and react for 2-24 hours under the conditions of pH 7.5-8.5 and temperature 20-40℃. d. Post-processing: After the reaction is completed, the substrate material is removed and then washed and dried in sequence to obtain the composite coating of the anti-Cronobacter biofilm on the surface of the substrate material.

4. The method for preparing the composite coating for the anti-Cronobacter biofilm according to claim 3, characterized in that, In step a, the buffer solution is a Tris-HCl buffer solution with a concentration of 10 mM and a pH of 8.

5.

5. The preparation method according to claim 3, characterized in that, The concentration of dopamine hydrochloride is 0.5-2.5 mg / mL; the concentration of chitosan oligosaccharide is 2.5-5.0 mg / mL.

6. The method for preparing the composite coating of the anti-Cronobacter biofilm according to claim 5, characterized in that, In step a, the mass ratio of dopamine hydrochloride to chitosan oligosaccharide is (1-2):(2-5).

7. The method for preparing the composite coating for the anti-Cronobacter biofilm according to claim 3, characterized in that, In step b, the cleaning process involves sequentially immersing the substrate material in acetone, ethanol, and deionized water for ultrasonic cleaning.

8. The method for preparing the composite coating for the anti-Cronobacter biofilm according to claim 3, characterized in that, In step c, the reaction is carried out under oscillation conditions; in step d, the drying temperature is 50°C and the drying time is 4-5 h.

9. The application of a composite coating for an anti-Cronobacter biofilm as described in claim 1 or 2, characterized in that, The composite coating is used to prepare products with anti-Cronobacter adhesion and / or anti-biofilm functions.

10. The application of the composite coating for anti-Cronobacter biofilm according to claim 9, characterized in that, The products include food processing machinery, food contact utensils, medical equipment, packaging materials, underwater cultural relic protection components, and underwater equipment.

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